1. Field of the Invention
Interest in transformation of crop and horticultural plants with added desirable agronomic traits is very high. Powerful new techniques for gene transfer recently have been developed for moving single genes and whole blocks of genes from one plant to another and even for moving genes from non-plants into plants. These new techniques are useful for generating specific plant genotypes and for the long term achievement of greater plant diversity through gene recombination. This invention relates to a novel method for introducing foreign genes into both monocotyledonous and dicotyledonous plants, thereby circumventing many of the limitations associated with the present-day technology in this field.
2. Description of the Prior Art
Several methods have been explored for introducing nucleic acid into a variety of cell types with the hope of developing a successful technique for the transformation of eukaryotic cells of plants and animals. A review of these methods is given by Potrykus [Biotechnology 8:535-541 (1990)].
Early approaches to transforming plants and animals focused on protoplast fusion [W. Schaffner, Proceedings National Academy Science U.S.A. 77:2163-2167 (1980); M. Rassoulzadegan et al., Nature 295:257-259 (1982)] and calcium phosphate coprecipitation with DNA or recombinant bacteriophage [F. L. Graham et al., Virology 52:456-467 (1973); M. Ishiura et al., Molecular Cell Biology 2:607-616 (1982)]. More recently, electroporation has been successful in introducing DNA into plant protoplasts [M. Fromm et al., Proceedings National Academy Science U.S.A. 82:5824-5828 (1985); M. Fromm et al., Nature 319:791-793 (1986)], fibroblasts [H. Liang et al., Biotechniques 6:550-558 (1988)], and mammalian red blood cells [T. Y. Tsong et al., Bibliographical Haematology 51:108-114 (1985); G. Chu et al., Nuclear Acids Research 15:1311-1326 (1987)].
Another approach which has been thoroughly investigated involves the use of Agrobacterium tumefaciens, and its tumor-inducing (Ti) plasmid. The use of vital vectors has also been considered [N. Brisson et al., Nature 310:511-514 (1984)].
Physical methods of introducing nucleic acid into cells include biolistic delivery IT. M. Klein et al., Bio/Technology 6:559-536 (1988); D. E. McCabe et al., Bio/Technology 6:923-926 (1988); and P. Christou et al., Plant Physiology 87:671-674 (1988)], Microinjection [G. Neuhaus et al., Theoretical Applied Genetics 75:30-36 (1987)], and infusion of cells physically disrupted with glass beads [Constanzo et al., Genetics 120:667-670 (1988)] or mineral fibers [Appel et al., Proc. Natl. Acad. Sci. U.S.A. 85:7670-7674 (1988); Kaeppler et al., Plant Cell Reports 9:415-418 (1990)].
There are several drawbacks to the aforementioned methods of DNA transformation in plants. Any of the techniques which uses or treats protoplasts is limited by the fact that protoplasts from many plant species are recalcitrant to regeneration into mature genetically stable plants. Accordingly, many of the most important economic crop plants have never been regenerated from protoplasts. In most cases where regeneration has been achieved, the production of plants from transformed protoplasts involves lengthy sterile culture and regeneration procedures.
The success of the electroporation method is dependent, in part, on optimizing parameters relative to the membrane, the DNA, and the electric field. Evidence for the success of transformation after electroporation has been measured by incorporation of radioactively labeled DNA [Tsong et al., supra], transient gene expression [H. Potter et al., Proceedings National Academy Science U.S.A. 81:7161-7165 (1984); O. Smithies et al., Nature 317:230-234 (1985)], and the formation of stable transformants [C. D. Riggs et al., Proceedings National Academy Science U.S.A. 83:5602-5606 (1986); H. Stopper et al., Z. Naturforsch. 40:929-932 (1985)]. Application of electroporation to cells and tissues has not been successful for generating transgenie clones, particularly in plants. Gene transfer by electroporation of protoplasts has met with some success, but the aforementioned problems of plant regeneration still exist.
Transformation of the most important crop plants, such as the cereals and legumes, has proven difficult with Agrobacterium. Agrobacterium has been shown to attach to wheat callus cells but no stable transformation was obtained [P.J. Dale et al., John Innes Institute Annual Report (1988); K. Dehesh et al. Science 250:1397 (1990)]. The number of plant species that are infected by this system is extremely limited, and other bacterial vectors are not currently available. Plant viruses are not known to integrate into the host genome and therefore show limited potential as vectors for stable transformation.
At present, the physical approaches seem to hold the most promise for genetic modification of cereals. However, the frequency of transient and integrative events with these techniques tends to be low. In plants, there is considerable difficulty in targeting cells that are competent for regeneration and/or integrative transformation. The meristem in cereals is well-protected, and it is unsettled whether any of the meristematic cells are competent for integrative transformation.